Induction of protein citrullination and auto-antibodies production in murine exposed to nickel nanomaterials.

Citrullination, or the post-translational deimination of polypeptide-bound arginine, is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. Recent studies have shown that nanomaterials can trigger protein citrullination, which might constitute a common pathogenic link to disease development. Here we demonstrated auto-antibody production in serum of nanomaterials-treated mice. Citrullination-associated phenomena and PAD levels were found to be elevated in nanomaterials -treated cell lines as well as in the spleen, kidneys and lymph nodes of mice, suggesting a systemic response to nanomaterials injection, and validated in human pleural and pericardial malignant mesothelioma (MM) samples. The observed systemic responses in mice exposed to nanomaterials support the evidence linking exposure to environmental factors with the development of autoimmunity responses and reinforces the need for comprehensive safety screening of nanomaterials. Furthermore, these nanomaterials induce pathological processes that mimic those observed in Pleural MM, and therefore require further investigations into their carcinogenicity.

Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a (Ink4a/Arf).

Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard.

Role of B Cell-Activating Factor in Chronic Obstructive Pulmonary Disease.

RATIONALE: B cell-activating factor (BAFF) plays a major role in activation of B cells and in adaptive humoral immune responses. In chronic obstructive pulmonary disease (COPD), lymphoid follicles have been associated with disease severity, and overexpression of BAFF has been demonstrated within lymphoid follicles of patients with severe COPD. OBJECTIVES: To investigate expression and localization of BAFF in the lungs of patients with COPD and to study the role of BAFF in COPD by antagonizing BAFF in a mouse model of chronic cigarette smoke (CS) exposure. METHODS: We quantified and localized BAFF expression in lungs of never-smokers, smokers without COPD, and patients with COPD and in lungs of air- or CS-exposed mice by reverse-transcriptase polymerase chain reaction, ELISA, immunohistochemistry, and confocal imaging. Next, to investigate the role of BAFF in COPD, we antagonized BAFF by prophylactic or therapeutic administration of a soluble fusion protein of the BAFF-receptor, BAFFR-Fc, in mice exposed to air or CS for 24 weeks and evaluated several hallmarks of COPD and polarization of lung macrophages. MEASUREMENTS AND MAIN RESULTS: BAFF expression was significantly increased in lungs of patients with COPD and CS-exposed mice. BAFF staining in lymphoid follicles was observed around B cells, CD4(+) cells, dendritic cells, follicular dendritic cells, and fibroblastic reticular cells. Prophylactic and therapeutic administration of BAFFR-Fc in mice reduced pulmonary B-cell numbers and prevented CS-induced formation of lymphoid follicles and increases in immunoglobulin levels. Interestingly, prophylactic BAFFR-Fc administration significantly attenuated pulmonary inflammation and destruction of alveolar walls. Moreover, antagonizing BAFF altered the phenotype of alveolar and interstitial macrophages. CONCLUSIONS: BAFF is significantly increased in lungs of patients with COPD and is present around both immune and stromal cells within lymphoid follicles. Antagonizing BAFF in CS-exposed mice attenuates pulmonary inflammation and alveolar destruction.

Minimal oxidation and inflammogenicity of pristine graphene with residence in the lung.

Two-dimensional graphitic carbon, graphene, is a new form of nanomaterial with great potential in a wide variety of applications. It is therefore crucial to investigate the behaviour of graphene in biological systems to assess potential adverse effects that might follow from inhalation exposure. In this study we focussed on medium-term effects of graphene in lung tissue by investigating the pulmonary inflammation 6 weeks after pharyngeal aspiration of unoxidised multilayered graphene platelets (GPs) in mice and assessed their biopersistence in the lung tissue using Raman spectroscopy. Additionally, GP degradation in vitro was examined after horseradish peroxidase (HRP) treatment up to 1 week. Building on our previous report showing acute inflammation in mice lungs at 1 day, pristine GP showed minimal inflammation in mouse lungs after 6 weeks even though no degradation of GP in lung tissue was observed and large deposits of GP were evident in the lungs. Raman analysis of GP in tissue sections showed minimal oxidation, and in vitro examinations of enzymatic oxidation of GP via HRP and H2O2 showed only slight increases in ID/IG ratio and the appearance of the Raman D' band at 1620 cm(-1) (surrogates of graphene oxidation). Our results showing non-inflammogenicity at medium time points have important implications in the hazard identification of GPs following inhalation exposure and for their use in biomedical applications. Additionally, the biopersistence of pristine GP in vivo with no associated inflammation could open the way to applications in tissue engineering and drug delivery.

Pulmonary toxicity of carbon nanotubes and asbestos - similarities and differences.

Carbon nanotubes are a valuable industrial product but there is potential for human pulmonary exposure during production and their fibrous shape raises the possibility that they may have effects like asbestos, which caused a worldwide pandemic of disease in the20th century that continues into present. CNT may exist as fibres or as more compact particles and the asbestos-type hazard only pertains to the fibrous forms of CNT. Exposure to asbestos causes asbestosis, bronchogenic carcinoma, mesothelioma, pleural fibrosis and pleural plaques indicating that both the lungs and the pleura are targets. The fibre pathogenicity paradigm was developed in the 1970s-80s and has a robust structure/toxicity relationship that enables the prediction of the pathogenicity of fibres depending on their length, thickness and biopersistence. Fibres that are sufficiently long and biopersistent and that deposit in the lungs can cause oxidative stress and inflammation. They may also translocate to the pleura where they can be retained depending on their length, and where they cause inflammation and oxidative stress in the pleural tissues. These pathobiological processes culminate in pathologic change - fibroplasia and neoplasia in the lungs and the pleura. There may also be direct genotoxic effects of fibres on epithelial cells and mesothelium, contributing to neoplasia. CNT show some of the properties of asbestos and other types of fibre in producing these types of effects and more research is needed. In terms of the molecular pathways involved in the interaction of long biopersistent fibres with target tissue the events leading to mesothelioma have been a particular area of interest. A variety of kinase pathways important in proliferation are activated by asbestos leading to pre-malignant states and investigations are under way to determine whether fibrous CNT also affects these molecular pathways. Current research suggests that fibrous CNT can elicit effects similar to asbestos but more research is needed to determine whether they, or other nanofibres, can cause fibrosis and cancer in the long term.

The biologically effective dose in inhalation nanotoxicology.

In all branches of toxicology, the biologically effective dose (BED) is the fraction of the total dose of a toxin that actually drives any toxic effect. Knowledge of the BED has a number of applications including in building structure-activity relationships, the selection of metrics, the design of safe particles, and the determination of when a nanoparticle (NP) can be considered to be "new" for regulatory purposes. In particle toxicology, we define the BED as "the entity within any dose of particles in tissue that drives a critical pathophysiogically relevant form of toxicity (e.g., oxidative stress, inflammation, genotoxicity, or proliferation) or a process that leads to it." In conventional chemical toxicology, researchers generally use the mass as the metric to describe dose (such as mass per unit tissue or cells in culture) because of its convenience. Concentration, calculated from mass, may also figure in any description of dose. In the case of a nanoparticle dose, researchers use either the mass or the surface area. The mass of nanoparticles is not the only driver of their activity: the surfaces of insoluble particles interact with biological systems, and soluble nanoparticles can release factors that interact with these systems. Nanoparticle shape can modify activity. In this Account, we describe the current knowledge of the BED as it pertains to different NP types. Soluble toxins released by NPs represent one potential indicator of BED for wholly or partially soluble NPs composed of copper or zinc. Rapid dissolution of these NPs into their toxic ions in the acidic environment of the macrophage phagolysosome causes those ions to accumulate, which leads to lysosome destabilization and inflammation. In contrast, soluble NPs that release low toxicity ions, such as magnesium oxide NPs, are not inflammogenic. For insoluble NPs, ζ potential can serve as a BED measurement because the exposure of the particle surface to the acidic milieu of the phagolysosome and interactions with the lysosomal membrane can compromise the integrity of the NPs. Researchers have explored oxidative potential of NPs most extensively as an indicator of the BED: the ability of an NP to cause oxidative stress in cells is a key factor in determining cell toxicity, inflammogenicity, and oxidative DNA adduct formation. Finally we discuss BEDs for high aspect ratio nanoparticles because long fibers or nanoplatelets can cause inflammation and further effects. These consequences arise from the paradoxically small aerodynamic diameter of fibers or thin platelets. As a result, these NPs can deposit beyond the ciliated airways where their extended dimensions prevent them from being fully phagocytosed by macrophages, leading to frustrated phagocytosis. Although knowledge is accumulating on the BED for NPs, many questions and challenges remain in understanding and utilizing this important nanotoxicological parameter.

Use of silver nanowires to determine thresholds for fibre length-dependent pulmonary inflammation and inhibition of macrophage migration in vitro.

BACKGROUND: The objective of this study was to examine the threshold fibre length for the onset of pulmonary inflammation after aspiration exposure in mice to four different lengths of silver nanowires (AgNW). We further examined the effect of fibre length on macrophage locomotion in an in vitro wound healing assay. We hypothesised that exposure to longer fibres causes both increased inflammation and restricted mobility leading to impaired clearance of long fibres from the lower respiratory tract to the mucociliary escalator in vivo. METHODS: Nine week old female C57BL/6 strain mice were exposed to AgNW and controls via pharyngeal aspiration. The dose used in this study was equalised to fibre number and based on 50 µg/ mouse for AgNW(14). To examine macrophage migration in vitro a wound healing assay was used. An artificial wound was created in a confluent layer of bone marrow derived macrophages by scraping with a pipette tip and the number of cells migrating into the wound was monitored microscopically. The dose was equalised for fibre number and based on 2.5 µg/cm(2) for AgNW(14). RESULTS: Aspiration of AgNW resulted in a length dependent inflammatory response in the lungs with threshold at a fibre length of 14 µm. Shorter fibres including 3, 5 and 10 µm elicited no significant inflammation. Macrophage locomotion was also restricted in a length dependent manner whereby AgNW in the length of ≥5 µm resulted in impaired motility in the wound closure assay. CONCLUSION: We demonstrated a 14 µm cut-off length for fibre-induced pulmonary inflammation after aspiration exposure and an in vitro threshold for inhibition of macrophage locomotion of 5 µm. We previously reported a threshold length of 5 µm for fibre-induced pleural inflammation. This difference in pulmonary and pleural fibre- induced inflammation may be explained by differences in clearance mechanism of deposited fibres from the airspaces compared to the pleural space. Inhibition of macrophage migration at long fibre lengths could account for their well-documented long term retention in the lungs compared to short fibres. Knowledge of the threshold length for acute pulmonary inflammation contributes to hazard identification of nanofibres.

Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo.

BACKGROUND: Frustrated phagocytosis has been stated as an important factor in the initiation of an inflammatory response after fibre exposure. The length of fibrous structures has been linked to the potential of fibres to induce adverse health effects for at least 40 years. However, we only recently reported for the first time the threshold length for fibre-induced inflammation in the pleural space and we implicated frustrated phagocytosis in the pro-inflammatory effects of long fibres. This study extends the examination of the threshold value for frustrated phagocytosis using well-defined length classes of silver nanowires (AgNW) ranging from 3-28 µm and describes in detail the morphology of frustrated phagocytosis using a novel technique and also describes compartmentalisation of fibres in the pleural space. METHODS: A novel technique, backscatter scanning electron microscopy (BSE) was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. A human monocyte-derived macrophage cell line THP-1 was used to investigate cell-nanowire interaction in vitro and the parietal pleura, the site of fibre retention after inhalation exposure was chosen to visualise the cell- fibre interaction in vivo after direct pleural installation of AgNWs. RESULTS: The length cut-off value for frustrated phagocytosis differs in vitro and in vivo. While in vitro frustrated phagocytosis could be observed with fibres≥14 µm, in vivo studies showed incomplete uptake at a fibre length of ≥10 µm. Recently we showed that inflammation in the pleural space after intrapleural injection of the same nanofibre panel occurs at a length of ≥5 µm. This onset of inflammation does not correlate with the onset of frustrated phagocytosis as shown in this study, leading to the conclusion that intermediate length fibres fully enclosed within macrophages as well as frustrated phagocytosis are associated with a pro-inflammatory state in the pleural space. We further showed that fibres compartmentalise in the mesothelial cells at the parietal pleura as well as in inflammatory cells in the pleural space. CONCLUSION: BSE is a useful way to clearly distinguish between fibres that are, or are not, membrane-bounded. Using this method we were able to show differences in the threshold length at which frustrated phagocytosis occurred between in vitro and in vivo models. Visualising nanowires in the pleura demonstrated at least 2 compartments--in leukocyte aggregations and in the mesothelium--which may have consequences for long term pathology in the pleural space including mesothelioma.

The threshold length for fiber-induced acute pleural inflammation: shedding light on the early events in asbestos-induced mesothelioma.

Suspicion has been raised that high aspect ratio nanoparticles or nanofibers might possess asbestos-like pathogenicity. The pleural space is a specific target for disease in individuals exposed to asbestos and by implication of nanofibers. Pleural effects of fibers depends on fiber length, but the key threshold length beyond which adverse effects occur has never been identified till now because all asbestos and vitreous fiber samples are heterogeneously distributed in their length. Nanotechnology advantageously allows for highly defined length distribution of synthetically engineered fibers that enable for in-depth investigation of this threshold length. We utilized the ability to prepare silver nanofibers of five defined length classes to demonstrate a threshold fiber length for acute pleural inflammation. Nickel nanofibers and carbon nanotubes were then used to strengthen the relationship between fiber length and pleural inflammation. A method of intrapleural injection of nanofibers in female C57Bl/6 strain mice was used to deliver the fiber dose, and we then assessed the acute pleural inflammatory response. Chest wall sections were examined by light and scanning electron microscopy to identify areas of lesion; furthermore, cell-nanowires interaction on the mesothelial surface of the parietal pleura in vivo was investigated. Our results showed a clear threshold effect, demonstrating that fibers beyond 4 µm in length are pathogenic to the pleura. The identification of the threshold length for nanofiber-induced pathogenicity in the pleura has important implications for understanding the structure-toxicity relationship for asbestos-induced mesothelioma and consequent risk assessment with the aim to contribute to the engineering of synthetic nanofibers by the adoption of a benign-by-design approach.

The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells.

Carbon nanotubes (CNT) are high aspect ratio nanoparticles with diameters in the nanometre range but lengths extending up to hundreds of microns. The structural similarities between CNT and asbestos have raised concern that they may pose a similar inhalation hazard. Recently CNT have been shown to elicit a length-dependent, asbestos-like inflammatory response in the pleural cavity of mice, where long fibres caused inflammation but short fibres did not. However the cellular mechanisms governing this response have yet to be elucidated. This study examined the in vitro effects of a range of CNT for their ability to stimulate the release of the acute phase cytokines; IL-1ß, TNFα, IL-6 and the chemokine, IL-8 from both Met5a mesothelial cells and THP-1 macrophages. Results showed that direct exposure to CNT resulted in significant cytokine release from the macrophages but not mesothelial cells. This pro-inflammatory response was length dependent but modest and was shown to be a result of frustrated phagocytosis. Furthermore the indirect actions of the CNT were examined by treating the mesothelial cells with conditioned media from CNT-treated macrophages. This resulted in a dramatic amplification of the cytokine release from the mesothelial cells, a response which could be attenuated by inhibition of phagocytosis during the initial macrophage CNT treatments. We therefore hypothesise that long fibres elicit an inflammatory response in the pleural cavity via frustrated phagocytosis in pleural macrophages. The activated macrophages then stimulate an amplified pro-inflammatory cytokine response from the adjacent pleural mesothelial cells. This mechanism for producing a pro-inflammatory environment in the pleural space exposed to long CNT has implications for the general understanding of fibre-related pleural disease and design of safe nanofibres.

Graphene-based nanoplatelets: a new risk to the respiratory system as a consequence of their unusual aerodynamic properties.

Graphene is a new nanomaterial with unusual and useful physical and chemical properties. However, in the form of nanoplatelets this new, emerging material could pose unusual risks to the respiratory system after inhalation exposure. The graphene-based nanoplatelets used in this study are commercially available and consist of several sheets of graphene (few-layer graphene). We first derived the respirability of graphene nanoplatelets (GP) from the basic principles of the aerodynamic behavior of plate-shaped particles which allowed us to calculate their aerodynamic diameter. This showed that the nanoplatelets, which were up to 25 µm in diameter, were respirable and so would deposit beyond the ciliated airways following inhalation. We therefore utilized models of pharyngeal aspiration and direct intrapleural installation of GP, as well as an in vitro model, to assess their inflammatory potential. These large but respirable GP were inflammogenic in both the lung and the pleural space. MIP-1α, MCP-1, MIP-2, IL-8, and IL-1ß expression in the BAL, the pleural lavage, and cell culture supernatant from THP-1 macrophages were increased with GP exposure compared to controls but not with nanoparticulate carbon black (CB). In vitro, macrophages exposed to GP showed expression of IL-1ß. This study highlights the importance of nanoplatelet form as a driver for in vivo and in vitro inflammogenicity by virtue of their respirable aerodynamic diameter, despite a considerable 2-dimensional size which leads to frustrated phagocytosis when they deposit in the distal lungs and macrophages attempt to phagocytose them. Our data suggest that nanoplatelets pose a novel nanohazard and structure-toxicity relationship in nanoparticle toxicology.

Identifying the pulmonary hazard of high aspect ratio nanoparticles to enable their safety-by-design.

High aspect ratio, or fiber-shaped, nanoparticles (HARNs) represent a growth area in nanotechnology as their useful properties become more apparent. Carbon nanotubes, the best known and studied of the HARNs are handled on an increasingly large scale, with subsequent potential for human inhalation exposure. Their resemblance to asbestos fibers precipitated fears that they might show the same type of pathology as that caused by asbestos and there is emerging evidence to support this possibility. The large number of other HARNs, including nanorods, nanowires and other nanofibers, require similar toxicological scrutiny. In this article we describe the unusual hazard associated with fibers, with special reference to asbestos, and address the features of fibers that dictate their pathogenicity as developed in the fiber pathogenicity paradigm. This paradigm is a robust structure:toxicity model that identifies thin, long, biopersistent fibers as the effective dose for fiber-type pathogenic effects. It is likely that HARNs will in general conform to the paradigm and such an understanding of the features that make fibers pathogenic should enable us to design safer HARNs.

Differential expression of microRNA 181b and microRNA 21 in hyperplastic polyps and sessile serrated adenomas of the colon.

This study was designed to analyse the potential diagnostic value of miR-181b and miR-21 for discriminating hyperplastic polyps (HP) from sessile serrated adenomas (SSA) without cytologic dysplasia. Using real-time polymerase chain reaction expression levels of miR-181b and miR-21 in 18 HPs, 19 SSAs without cytologic dysplasia and 20 normal colonic mucosal specimens were examined. In addition, 20 colorectal cancers specimen were analysed for miR-181b expression. Data were normalised to RNU48 as an internal control. A differential expression of miR-181b and miR-21 was found in HPs, SSAs, and normal colonic mucosa with highest expression levels in SSAs. Levels of miR-181b but not miR-21 differed in HPs and normal mucosa. SSAs exhibited both significantly higher miR-181b levels (up to 2.01-fold; P < 0.001) and miR-21 levels (up to 1.82-fold; P = 0.011) than HPs. In contrast to HPs, SSAs are characterised by high levels of miR-181b and miR-21 expression. However, due to the overlap of values, miR-181b and miR-21 evaluation did not allow discrimination of the two lesions in every case.